Tetanus toxin is a protein produced by Clostridium tetani, an anaerobic bacterium whose spores are commonly found in soil and animal waste. Tetanus toxin is an extremely potent neurotoxin which causes tetanus, a potentially fatal condition that affects the nervous system and is characterized by painful, uncontrolled muscle contractions. It is synthesized as a single polypeptide and is post-translationally modified to provide light and heavy chains linked by disulfide bonds.
The structure of tetanus toxin has been cloned and sequenced (Fairweather et al. (1986), J. Bacteriol. 165; 21-27). It is a 150 kD protein comprising 1315 amino acids. Papain digestion of the tetanus toxin results in the cleavage of the heavy chain to give two fragments, fragment B and C. Fragment C (also termed herein as tetanus toxin fragment C or “TTC”) is a 50 kD polypeptide at the C-terminus which is capable of protecting mice against lethal challenge with tetanus toxin. Purified TTC is nontoxic in animals. Expression of Fragment C was carried out in E. coli, where it was fused to part of the E. coli trpE protein or to fragment B of tetanus toxin (Fairweather et al. (1987), Infection and Immunity 55: 2541-2545).
Other researchers have shown that TTC protective ability against tetanus can be successfully expressed in Salmonella typhimurium (Chatfield et al. (1992), Biotechnology, 10(8): 888-92). Salmonella typhi. vaccine strain CVD908, developed as a live oral typhoid vaccine has been used to deliver heterologous vaccine antigens. Specifically, the plasmid pTET containing the S1 subunit of pertussis toxin fused to fragment C was expressed at high levels in CVD908 when it is driven by the nirB promoter (Barry et al. (1996), Infection and Immunity 64(10): 4172-4181).
The adjuvant effect of TTC was first found when it was co-administered with the full length P28 glutathione S-transferase (GST) protein of S. mansoni. As disclosed in WO 94/03615 or WO 95/04151, expression of GST was increased when it is fused to TTC. It is taught that TTC acts as a carrier protein that “rescues” the expression of otherwise difficult to express proteins such as GST—a so-called “expression rescue” (see Gomez-Duarte et al. (1995), Vaccine 13:1596-1602 and Khan et al. (1994), Proc. Natl. Acad. Sci. USA 91:11261-11265). Because of the “expression rescue”, it seems reasonable that the antigen titer against GST increases, since more GST or a form of the GST protein which is accessible for the immune system is produced. This assumption is corroborated by the finding that more copies of a TTC-GST fusion gene lead to an increased expression (see Example 5 of WO 94/03615). However, since TTC is a bacterial protein that was long ago applied as antigen to affect a protective immune response against Clostridium tetani (Boucher et al. (1994), Inf. Immunity 62(2):449-456), it is not surprising that the antibody titer is increased when another bacterial host cell (Salmonella sp.) of the same family (enterobacteriaceae) is used to express GST (Lee et al. (2000), Inf. Immunity 68(5)2503-2512). Lee et al. therefore conclude that TTC may be exploited as a potent tool to immunostimulate the production of antibodies directed against antigens in live vaccines, i.e., in living bacteria. In fact, the likelihood that the expression of a bacterial protein of one species in a bacterial host cell of another species is successful is unequally higher than expression of the bacterial protein in a non-bacterial host cell such as a virus.
As mentioned above, the phenomenon of “expression rescue” is only known for bacteria. In recombinant viruses, in particular poxviruses, applied as carriers for the expression of an antigen against which an immune response is desired, such a phenomenon is not known: Those of skill in the art use either multiple copies of the antigen or strong promoters that confer high expression of the antigen in order to “offer” an excess antigen to the immune system with the aim of affecting an immune response. Also, thus far, it was neither known nor believed to be possible to increase an immune response against an antigen, in particular against a bacterial antigen, by way of co-expressing the antigen together with a bacterial protein or even as a fusion protein with the bacterial protein when included in a recombinant virus. In fact, thus far, neither problems nor difficulties with the expression of an antigen in a virus acting as carrier for an antigen were discussed in the prior art, nor were difficulties known in obtaining a sufficient immune response against an antigen when a poxvirus was used as carrier to express the antigen.
Thus, while it was known in the art that a bacterial protein could “rescue” the expression of a an otherwise difficult to express antigenic protein in bacterial host cells, whereby an immune response against the antigenic protein could be enhanced, up to the present invention, any issue such as the expression of an otherwise difficult to express protein or the increment of the antibody titer against a desired antigen protein, was neither recognized nor known in the art when a poxvirus was used as carrier and which would thus have required improvement.
TTC is also known to contain T helper cell epitopes. These epitopes are exploited in that less immunogenic proteins are equipped with such epitopes with the aim of rendering them more immunogenic. Such an approach is, for example, described in WO 2008/045346: Specifically, the breast cancer antigen Her2/neu is modified by substituting amino acids to resemble a T cell epitope from TTC.
Another approach for rendering a cancer antigen more immunogenic is the fusion between TTC and DR4 or DR5. DR4 and DR5 are also known as TRAIL-R1 and TRAIL R2, both of which are capable of transducing an apoptotic signal to a cell expressing them. The idea described in WO 2010/054156 is to render DR4 and DR5 more immunogenic if fused to TTC.
Just another approach to render viral antigens more immunogenic is briefly envisaged in WO 92/22641: In particular, in passing by, this application suggests a co-expression approach of an HIV protein and TTC. However, apart from a mere hypothetical description, this application fails to provide any data on the suggested approach.
The present invention provides aspects and embodiments concerning recombinant poxvirus vectors comprising a TTC coding sequence, wherein said TTC coding sequence is operably linked to a coding sequence encoding a bacterial antigenic determinant. The present invention also provides methods and uses applying these recombinant poxvirus vectors in the treatment of subjects which would benefit from the administration of said recombinant poxvirus vectors. These aspects and embodiments are characterized and described herein, illustrated in the Examples, and reflected in the claims.